Apo AI/CIII genomic polymorphisms predictive of atherosclerosis

• US 4,801,531 A
• Filed: 09/30/1985
• Issued: 01/31/1989
• Est. Priority Date: 04/17/1985
• Status: Expired due to Fees

• Current Assignee: California Biotechnology, Inc. (Johnson & Johnson)
• Original Assignee: Biotechnology Research Partners, Ltd.
• Inventors: Frossard, Philippe M.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Wagner, Richard
• Application Number:
  US06/782,666
• Time in Patent Office: 1,219 Days
• Field of Search: 435/6, 435/810, 436/63, 436/94, 436/501, 436/503, 536/27, 935/11, 935/78, 935/79
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/172 Haplotypes
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention offers an early detection method for atherosclerosis using genetic analysis to detect a polymorphisms shown to be correlated with this disease which are proximal to the apolipoprotein AI (apoAI) and aplipoprotein CIII (apoCIII) gene complex. All individuals with a 300 bp deletion 4 kb upstream of the apoAI gene are destined to experience severe atherosclerotic symptomologies. Individuals with a polymorphism 5.4 kb 5'"'"' of the apoAI gene or a PvuII polymorphism in the first intron of the apoCIII gene also seem to be at greater risk. A haplotype with MspI and XmnI/7.2 polymorphisms in this general region seem to be protected. Additional polymorphic sites in the DNA sequence associated with the apoAI/CIII gene complex provide a means for genetically fingerprinting individuals, and for identifying persons at risk with respect to disorders relating to lipid metabolism and transport.

Claims

1. A method for predicting the development of atherosclerosis in an individual human subject, which method comprises detecting the presence or absence of the "XmnI/8.2" polymorphism, which is a deletion of a 300 bp segment of DNA at 4 kb 5'"'"' of the apoAI gene.

2. The method of claim 1 which comprises detecting the presence of absence of a diagnostic length DNA fragment of human genomic DNA which has been digested with a restriction endonuclease, said fragment hybridizing to p5'"'"'AI probe, or to a substantially equivalent probe.

3. The method of claim 2 wherein the restriction endonuclease is XmnI and the length of the fragment is 8.2 kb.

4. The method of claim 2 wherein the restriction endonuclease is ApaI and the length of the restriction fragment is 3.2 kb.

5. The method of claim 2 wherein the restriction endonuclease is BglI and the length of the fragment is 5.9 kb.

6. The method of claim 2 wherein the restriction endonuclease is BstEII and the length of the fragment is 2.1 kb.

7. The method of claim 2 wherein the restriction endonuclease is MspI and the length of the fragment is 5.9 kb.

8. The method of claim 2 wherein the restriction endonuclease is PvuII and the length of the fragment is 1.6 kb.

9. The method of claim 2 wherein the restriction endonuclease is RsaI and the length of the fragment is 3.2 kb.

10. The method of claim 2 wherein the restriction endonuclease is StuI and the length of the fragment is 5.5 kb.

11. The method of claim 2 wherein the restriction endonuclease is TaqI and the length of the fragment is 9.1 kb.

12. A method for predicting the development of atherosclerosis in an individual human, which method comprises detecting the presence or absence of the "ApaI" polymorphism which is 5.4 kb 5'"'"' of the apoAI gene.

13. The method of claim 12 which comprises detecting the presence of absence of a 2.2 kb DNA fragment of human genome which has been digested with ApaI, said fragment hybridizing with p5'"'"'AI probe or a substantially equivalent probe.

14. A method for predicting the development of atherosclerosis in an individual human, which method comprises detecting the presence of absence of the "PvuII" polymorphism which is in the first intron of the apoCIII gene.

15. The method of claim 15 which comprises detecting the presence or absence of a 0.87 kb DNA fragment of human genome which has been digested with PvuII, said fragment hybridizing with apoCIII probe or a substantially equivalent probe.

16. A method for predicting decreased susceptibility to atherosclerosis in an individual human which method comprises detecting the presence or absence of the "XmnI/7.2" polymorphism which is 3.7 kb 5'"'"' of the apoAI gene and the "MspI" polymorphism which is in the third intron of the apoAI gene.

17. The method of claim 16 which comprises detecting the presence or absence of a 7.2 kb DNA fragment of human genome which has been digested with XmnI, said fragment hybridizing with apoAI or p5'"'"'AI probe or a substantially equivalent probe and a 1.75 kb DNA fragment of human genome digested with MspI, said fragment hybridizing with apoAI probe or a substantially equivalent probe.

18. A method for ascertaining individual human subjects having a propensity for disorders relating to lipid transport and metabolism, which method comprises detecting the presence or absence of one or more polymorphisms selected from the group consisting of:

the "ApaI" polymorphism 5.4 kb 5'"'"' of the apoAI gene;

the "XmnI/8.2" polymorphism 4 kb 5'"'"' of the apoAI gene;

the "XmnI/7.2" polymorphism 3.7 kb 5'"'"' of the apoAI gene in combination with the "MspI" polymorphism in the 3rd intron of the apoAI gene; and

the "PvuII" polymorphism in the 1st intron of the apoCIII gene.

19. The method of claim 18 wherein each of said polymorphisms is detected in total human genomic DNA by, respectively,

digestion with ApaI and detection of a 2.2 kb fragment using p5'"'"'AI probe or a substantial equivalent;

digestion with XmnI and detection of an 8.2 kb fragment using p5'"'"'AI or a substantial equivalent or apoAI or a substantial equivalent;

digestion with XmnI and detection of a 7.2 kb fragment using p5'"'"'AI or a substantial equivalent or apoAI or a substantial equivalent in combination with digestion with MspI and detection of 1.75 kb fragment using apoAI probe or a substantial equivalent; and

digestion with PvuII and detection of a 0.87 kb fragment using apoCIII probe or a substantial equivalent.

20. A method of genetically identifying an individual, which method comprises assessing the presence or absence of at least one polymorphism by

contacting a genomic DNA digest with apoAI probe or a substantial equivalent, wherein said digest is obtained by digesting the genome with an enzyme selected from the group consisting of XmnI, HgiAI, and PstI, or a subset thereof, and

detecting the presence or absence of a diagnostic length DNA fragment which hybridizes to said probe.

21. A method of genetically identifying an individual, which method comprises assessing the presence or absence of at least one polymorphism by

contacting a genomic DNA digest with p5'"'"'AI probe or a substantial equivalent, wherein said digest is obtained by digesting the genome with an enzyme selected from the group consisting of ApaI, XmnI, BglI, BstEII, PvuII, RsaI, StuI and TaqI or a subset thereof, and

detecting the presence of absence of a diagnostic length DNA fragment which hybridizes to said probe.

22. A method of genetically identifying an individual which method comprises assessing the presence or absence of at least one polymorphism by

contacting a genomic DNA digest with apoCIII probe or a substantial equivalent wherein said digest is obtained by digesting the genome with BanII, PvuII or both.

23. The method of claim 2 wherein the restriction endonuclease is selected from the group consisting of XmnI, ApaI, BglI, BstEII, MspI, PvuII, RsaI, StuI, and TagI and the length of the fragment is, respectively, 8.2 kb, 3.2 kb, 5.9 kb, 2.1 kb, 5.9 kb, 1.6 kb, 3.2 kb, 5.5 kb, 9.1 kb.

24. A method of genetically identifying an individual, which method comprises detecting the presence or absence of one or more polymorphisms selected from the group consisting of:

the "ApaI" polymorphism 5.4 kb 5'"'"' of the apoAI gene;

the "XmnI/8.2" polymorphism 4 kb 5'"'"' of the apoAI gene;

the "XmnI/7.2" polymorphism 3.7 kb 5'"'"' of the apoAI gene in combination with the "MspI" polymorphism in the 3rd intron of the apoAI gene;

the "HgiAI" polymorphism in the 3rd exon of the apoAI gene;

the "PstI" polymorphism 0.3 kb 3'"'"' of the apoAI gene; and

the "PvuII" polymorphism in the 1st intron of the apoCIII gene.

25. The method of claim 24 wherein each said polymorphism is detected in total human genomic DNA by, respectively,

digestion with ApaI and detection of a 2.2 kb fragment using p5'"'"'AI probe or a substantial equivalent;

digestion with XmnI and detection of an 8.2 kb fragment using p5'"'"'AI or a substantial equivalent or apoAI or a substantial equivalent;

digestion with XmnI and detection of a 7.2 kb fragment using p5'"'"'AI or a substantial equivalent or apoAI or a substantial equivalent

in combination with digestion with MspI and detection of 1.75 kb fragment using apoAI probe or a substantial equivalent;

digestion with HgiAI and detection of 0.94 kb fragment using apoAI probe or a substantial equivalent;

digestion with PstI detection of a 3.2 kb fragment using apoAI probe or a substantial equivalent; and

digestion with PvuII and detection of a 0.87 kb fragment using apoCIII probe or a substantial equivalent.

26. A method to detect the SstI/BanII polymorphism in the 4th exon of the gene which comprises digesting the genome with BanII and detecting a 1.0 and 0.8 kb fragment using apoCIII probe or a substantial equivalent.

27. A reagent kit useful in performing the method of claim 1, which kit includes XmnI restriction enzyme and at least one probe selected from apoAI gene probe or a substantial equivalent and p5'"'"'AI probe or a substantial equivalent.

28. A reagent kit useful in performing the method of claim 1, which kit includes p5'"'"'AI probe or substantial equivalent and at least one restriction enzyme selected from the group consisting of ApaI, XmnI, BglI, BstEII, MspI, PvuII, RsaI, StuI, and TaqI.

29. A reagent kit useful in performing the method of claim 12, which kit includes ApaI restriction enzyme and p5'"'"'AI probe or a substantial equivalent.

30. A reagent kit useful in performing the method of claim 14, which kit includes PvuII restriction enzyme and apoCIII probe or a substantial equivalent.

31. A reagent kit useful in performing the method of claim 16, which kit includes XmnI restriction enzyme MspI restriction enzyme, and apoAI probe or a substantial equivalent.

32. A reagent kit useful in ascertaining individual human subjects having a propensity for disorders related to lipid transport and metabolism, which kit includes at least one probe selected from the group consisting of apoAI probe, p5'"'"'AI probe, and apoCIII probe, or their substantial equivalents, and at least one restriction enzyme selected from the group consisting of ApaI, XmnI, HgiAI, MspI, BanII, PvuII, BglI, BstEII, RsaI, StuI, and TaqI.